Laurane G. Mendelsohn

Multiple folate enzyme inhibition: mechanism of a novel pyrrolopyrimidine-based antifolate LY231514 (MTA)

Extensive biochemical and pharmacological evidence indicates that LY231514 is a novel antifolate antimetabolite. LY231514 is transported into cells mainly through the reduced folate carrier system and extensively metabolized to polyglutamated forms. The polyglutamates of LY231514 inhibit at least three key folate enzymes: TS, DHFR, and GARFT, and to a lesser extent AICARFT and C1-tetrahydrofolate synthase. The combined effects of the inhibition exerted by LY231514 at each target give rise to an unusual end-product reversal pattern at the cellular level that is distinct from those of other inhibitors such as methotrexate and the quinazoline antifolates. The metabolic effects exerted by LY231514 on the folate and nucleotide pools are also quite distinct from those of MTX and LY309887. The efficient polyglutamation, longer cellular retention and the multiple folate enzyme inhibition mechanism may all have contributed directly to the exciting antitumor responses now observed in Phase I and II studies. The multitargeted inhibition mechanism of LY231514 is particularly intriguing. This new level of mechanistic insight, which has evolved from the study of LY231514, challenges the traditional concept and paradigm of antifolate drug discovery and development which focused on developing very potent and selective inhibitors of single folate enzyme targets, such as DHFR, TS or one of the enzymes along the de novo purine biosynthetic pathway. Given the complex nature of folate metabolism and the critical role of folates in maintaining the physiological functions of living systems, it is completely reasonable to suspect that agents which can interfere at multiple sites in the folate pathway may trigger and cause more biochemical imbalance in the cellular DNA and RNA synthesis of malignant cells than agents that act on a single point (Fig. 5). In conclusion, LY231514 (MTA) is a new generation antifolate antimetabolite demonstrating inhibitory activity against multiple folate enzymes including TS, DHFR and GARFT. In current phase II studies, MTA is broadly active as a single agent and is showing very encouraging antitumor activity in multiple solid tumors including colorectal, breast and non-small cell lung cancers (38-43). The every three week dosing schedule has proven to be convenient and easy to administer and the clinical toxicities of LY231514 seem to be well tolerated. More advanced and extensive clinical trials of LY231514 are currently in progress.

Cell cycle effects of antifolate antimetabolites: implications for cytotoxicity and cytostasis

Abstract Purpose: Cell cycle-related events in CCRF-CEM lymphocytic leukemia cells were examined subsequent to inhibition of thymidylate synthase (TS) or GAR formyltransferase (GARFT) and prior to cell death or stasis. Methods: Cell populations were treated with the GARFT inhibitors 6R-5,10-dideazatetrahydrofolate (lometrexol) or LY309887, the TS inhibitor ZD1694, or the multitargeted antifolate LY231514. DNA content, nucleoside precursor incorporation and proliferating cell nuclear antigen (PCNA) expression as functions of drug treatment were assessed by multiparameter flow cytometry. Cellular respiration was measured by MTT analysis and apoptosis was detected by extraction of DNA fragments. Results: Cell populations treated for up to 96 h with lometrexol or LY309887 did not replicate and maintained a cell cycle distribution with distinct G1, S and G2/M regions. The number of S phase cells in treated populations was slightly elevated relative to control as measured by DNA content and PCNA. However, these cells were unable to incorporate 5-bromodeoxyuridine (BrdU). Throughout treatment, cells incubated with GARFT inhibitors maintained intact membranes and respired at a level comparable to untreated cells. In contrast, ZD1694 as well as LY231514, induced synchronization of the treatment population at the G1/S interface within 12 h of drug addition. This was followed by synchronous entry of the population into S phase. After 24 h of treatment, more than 90% of the cells were capable of incorporating BrdU and stained positive for PCNA. DNA fragmentation occurred in cells treated with ZD1694 or LY231514 but not in those treated with GARFT inhibitors. In addition, the viable cells remaining after 24–48 h of treatment with ZD1694 or LY231514 were respiring at twice the level of untreated cells. Conclusion: These results demonstrate that the distinct endpoints of GARFT and TS inhibition are preceded by distinct cell cycle and metabolic alterations.

Phencyclidine receptors in rat brain cortex

The binding of [3H]phencyclidine (PCP) to receptors in rat brain cortex has been studied. Two receptors have been detected, a high affinity receptor site with a KD of 23.5 +/- 7.4 nM and a low affinity site with a KD of 7.6 +/- 1.8 microM. The binding of [3H]PCP to its receptors was pH and temperature dependent and was destroyed by heat-denaturation. The binding of [3H]PCP was inhibited by compounds which produce PCP-like behavioral effects including dexoxadrol, etoxadrol and ketamine as well as a novel series of benz(f)isoquinolines. The low affinity site was blocked by PCP, etoxadrol and (+)-SKF-10,047 but not morphine or leu-enkephalin, suggesting that it also represents a specific PCP site. Stereoselective displacement of PCP at the high affinity receptor was observed with the isomers of cyclazocine, cyclorphan, SKF-10,047 and dioxadrol (dexoxadrol and levoxadrol). Naloxone, 4,5,6,7-tetrahydroisoxazolo(S,4-C)pyridin-3-ol (THIP) hydrate and haloperidol inhibited binding poorly (Ki greater than 1 microM), suggesting that these compounds do not interact significantly with the high affinity PCP receptor in vivo. The affinity of ligands for the phencyclidine receptor was highly correlated (r = 0.714, P less than 0.01) with their potency to produce catalepsy in pigeons.

The insulin-like growth factor-II (IGF-II) receptor of rat brain: regional distribution visualized by autoradiography.

The presence of insulin-like growth factor-II (IGF-II) in brain and cerebral spinal fluid prompted us to investigate the distribution of receptors for this peptide in rat brain slices. Human 125I-IGF-II (10 pM) was incubated for 16 h at 4 degrees C with thaw-mounted slices of rat brain from 11 different brain regions. Incubations in the absence or presence of excess unlabeled human IGF-II or insulin were performed and the labeled tissues were exposed to X-ray film for 4-7 days. Autoradiographs showed dense labeling in the granule layers of the olfactory bulbs, deep layers of the cerebral cortex, pineal gland, anterior pituitary, hippocampus (CA1-CA4, and dentate gyrus), and the granule cell layers of the cerebellum. Unlabeled IGF-II eliminated most of the binding in these brain regions while insulin produced only a minimal reduction in the amount of 125I-IGF-II bound. These results indicate that a neural receptor for IGF-II is uniquely distributed in rat brain tissue supporting the notion that this peptide might play an important role in neuronal functioning.

Biochemistry and pharmacology of glycinamide ribonucleotide formyltransferase inhibitors : LY309887 and lometrexol

SummaryLometrexol, a tight-binding antifolate inhibitor of the purine de novo enzyme glycinamide ribonucleotide formyltransferase (GARFT), was the first GARFT inhibitor to be investigated clinically. Unexpected observations of delayed cumulative toxicity prompted a search for a second generation antimetabolite with a more favorable biochemical, pharmacological and toxicological profile. LY309887, 6R-2′,5′-thienyl-5,10-dideazatetrahydrofolic acid, had 9-fold greater potency to inhibit GARFT (Ki = 6.5 nM) compared to lometrexol. Like lometrexol, LY309887 was activated by folypolyglutamate synthetase, however, it had a lower first order rate constant. In vitro and in vivo data were consistent with these observations: polyglutamation of LY309887 was less extensive compared to lometrexol and livers of mice accumulated fewer polyglutamates of LY309887 than polyglutamates of lometrexol. The affinities of these two compounds for isoforms of human folate receptors (FR) were compared. Lometrexol had a 6-fold higher affinity for FRα than LY309887 and both compounds had higher affinity for the α isoform compared to the β isoform. The selectivity of LY309887 for FRα (β(Ki)/α(Ki) = 10.5) was twice that of lometrexols (β/α = 5.0). Lometrexol and LY309887 were potent cytotoxic compounds against the human leukemia cell line CCRF-CEM with IC50s of 2.9 nM and 9.9 nM, respectively. In vivo, LY309887 was more potent than lometrexol at inhibiting tumor growth in the C3H mammary murine tumor model and several tumor xenografts. Excellent efficacy was achieved by both compounds in several colon xenografts. In two pancreatic human xenografts, LY309887 achieved greater efficacy than lometrexol. In summary, the biochemical and pharmacological properties of lometrexol and LY309887 support the hypothesis that these antifolates will have clinical activity against human solid tumors. LY309887 is a second generation GARFT inhibitor with biochemical and pharmacological properties which distinguish it from lometrexol and suggest that it will have broad antitumor activity, a different pharmacokinetic profile and produce less toxicity than lometrexol in cancer patients.

Enzymatic rationale and preclinical support for a potent protein kinase Cβ inhibitor in cancer therapy

The macrocyclic bisindolylmaleimide, LY333531, selectively inhibits protein kinase C beta 1 and beta 2 isoforms with an approximate IC50 of 5 nanomolar. The efficacy of LY333531 administered alone and in combination with cytotoxic cancer therapies in models of non-small cell lung carcinoma and brain tumors was determined in vivo. In the Lewis lung carcinoma, administration of LY333531 enhanced the activity of paclitaxel and fractionated radiation and, to a lesser degree, carboplatin and gemcitabine. In the human T98G glioblastoma multiforme xenograft, the addition of LY333531 to treatment with carmustine (BCNU) resulted in enhanced tumor response in a nodule grown subcutaneously and increased life-span in animals bearing an intracranial tumor from 37 days in the control animals to 64 days in the BCNU treated animals, and to 104 days in the LY333531 plus BCNU treated animals with 4 out of 5 animals being long-term survivors.

The role of dietary folate in modulation of folate receptor expression, folylpolyglutamate synthetase activity and the efficacy and toxicity of lometrexol

We have studied the molecular effects of a LFD in a murine model in order to better define the biochemical changes associated with folate deficiency. In addition, we have demonstrated the effect of a LFD on the pharmacokinetic profile and therapeutic activity and toxicity of lometrexol. These studies showed increased density of FR in tumors implanted in LFD mice and a decrease in the affinity of these receptors for folic acid. The results suggest that tumors can compensate for low folate bioavailability by up-regulation of a second FR with slightly lower affinity for folic acid. The higher density of this FR would provide greater capacity for garnering serum folate. FPGS activity increased in several tumors and liver and kidney of LFD mice. The increase in this enzyme activity would result in enhanced polyglutamation of folates and classical antifolates and thus increased cellular retention. Consistent with these changes in liver FPGS, mice injected i.v. with a single dose of lometrexol accumulated significantly more drug in liver and tumors of LFD animals compared to SD mice. Also, higher liver concentrations of lometrexol persisted longer in LFD mice. Polyglutamate analysis showed that longer polyglutamate forms appeared earlier in liver of LFD mice. After 7 days, longer polyglutamyl forms were recovered from liver of LFD mice (octa- and hepta-glutamyl lometrexol) compared to those on SD. A comparison of the efficacy and toxicity of lometrexol in C3H mammary tumor-bearing mice showed that in mice on LFD, lometrexol treatment produced a delayed toxicity with an LD50 of 0.1-0.3 mg/kg, a 3000-fold increase in lethality compared to SD mice. Supplementation of mice with folic acid restored anti-tumor activity and increased the therapeutic dose-range over which efficacy could be assessed. These studies support the use of folic acid supplementation for cancer patients treated with antifolate therapy in order to prevent the biochemical changes in FR and FPGS associated with folate deficiency, prevent delayed toxicity to GARFT inhibitors and enhance the therapeutic potential of this class of drugs.

Preclinical and Clinical Evaluation of the Glycinamide Ribonucleotide Formyltransferase Inhibitors Lometrexol and LY309887

The importance of the purine de novo pathway in providing DNA precursors for cancer cell growth led to the hypothesis that novel antifolate inhibitors of glycinamide ribonucleotide formyltransferase (GARFT), the first folate-dependent enzyme in this pathway, might have utility in the treatment of cancer. In 1987, clinical investigations were initiated with lometrexol (6R-dideazatetrahydrofolic acid, 6R-DDATHF), a novel “tight-binding” inhibitor of GARFT with potent antitumor activity in a number of murine and human xenograft solid tumors. Unexpected observations of delayed cumulative toxicity in phase I clinical trials prompted extensive preclinical investigations of the dynamics of folate status on the efficacy and toxicity of GARFT inhibitors and other antifolates (1). In addition, structure-activity studies have led to the identification of a second generation GARFT inhibitor, LY309887 (2´, 5´-thienyl-dideazatetrahydrofolic acid), which is more potent than lometrexol and has greater antitumor efficacy in vivo (2). Biochemical and pharmacological differences between LY309887 and lometrexol with respect to potency to inhibit GARFT, differential transport and storage in liver, and polyglutamation suggest that LY309887 may have greater antitumor efficacy and more manageable toxicity in the clinic than lometrexol. A murine model of the delayed cumulative toxicity seen with lometrexol has been refined and characterized to provide greater understanding of the pharmacokinetics and pharmacodynamics of these events. In concert with recently published nutritional data on the folate status of humans and more sophisticated methods of assessing and modulating antifolate toxicities through vitamin supplementation, antifolate therapy may be poised to enter a new phase of clinical success. In this report, we describe LY309887, a GARFT inhibitor with unique biochemical and pharmacological properties that has antitumor activity against a broad panel of human xenograft tumors, and greater potency than lometrexol both as an inhibitor of GARFT and as an inhibitor of tumor growth in vivo. An overview of the phase I clinical results with lometrexol and the design of the phase I clinical trial with LY309887 will be presented.

Dietary folate and folylpolyglutamate synthetase activity in normal and neoplastic murine tissues and human tumor xenografts.

The importance of polyglutamation for the activation of natural folates and classical antifolates and recent evidence for the role of dietary folate as a biochemical modulator of antifolate efficacy led us to investigate the influence of changes in dietary folate on folylpolyglutamate synthetase (FPGS) activity. Activities were measured using lometrexol (6R-5,10-dideazatetrahydrofolic acid) as a substrate for FPGS with extracts of murine tissues, murine tumors, and human tumor xenografts from mice on standard diet or low folate diet. Tissues and tumors from mice on standard diet exhibited a 6-fold range of FPGS activity. Kidney had the lowest activity (36 pmol/hr.mg protein), followed by the human xenograft PANC-1 pancreatic carcinoma (46 pmol/hr.mg protein), liver (109 pmol/hr.mg protein), murine C3H mammary tumor (112 pmol/hr.mg protein), and the human xenograft MX-1 mammary carcinoma (224 pmol/hr.mg protein). In response to restricted dietary folate, four out of five tissues had significantly increased (25-50%) FPGS activity. Only the tumor with highest FPGS activity under standard diet conditions (MX-1 mammary) did not respond to low folate diet. The results indicate that changes in dietary folate intake can modulate FPGS activity significantly in vivo and suggest that the tissue distribution and toxicities of classical antifolates requiring polyglutamation for activation and cellular retention will be influenced significantly by folate status of the host.

Opioid antinociceptive effects of delta-receptor antagonists

The antinociceptive effects of delta opioid receptor antagonists (ICI 154129 and ICI 174864) have been studied using the mouse writhing assay. When administered intracerebroventricularly (ICV), ICI 154129 and ICI 174864 produced dose-related inhibition of writhing with respective ED50s of 97 micrograms/mouse and 1.4 micrograms/mouse. Inhibition of writhing by ICI 174864 (3 micrograms, ICV) was antagonized by subcutaneous (SC) naloxone doses of 0.1 mg/kg and greater. Pretreatment of mice with 80 mg/kg (SC) of beta-funaltrexamine (beta-FNA), an irreversible mu-receptor antagonist, 28 hr before ICV injection of ICI 174864 shifted the dose-effect curve for ICI 174864 to the right (ED50 of 7.3 micrograms/mouse). When administered SC, ICI 174864 inhibited writhing with an ED50 of 8.5 mg/kg. Maximal inhibition occurred 30 min after SC administration and decreased 50% by 2 hr. After beta-FNA pretreatment, doses of ICI 174864 as high as 80 mg/kg (SC) did not inhibit writhing. There was no antinociceptive effect of ICI 174864 in mice chronically maintained on morphine, i.e., chronic morphine produced cross-tolerance to the delta antagonist. These results show that delta-selective receptor antagonists produced antinociception which was related to the mu-receptor, but was probably not a result of direct agonist action.